Potential roles of 5´ UTR and 3´ UTR regions in post-trans-criptional regulation of mouse Oct4 gene in BMSC and P19 cells

Document Type: Original Article

Authors

1 Department of Genetics, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran

2 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Abstract

Objective(s):OCT4 is a transcription factor required for pluripotency during early embryogenesis and the maintenance of identity of embryonic stem cells and pluripotent cells. Therefore, the effective expression regulation of this gene is highly critical. UTR regions are of great significance to gene regulation. In this study, we aimed to investigate the potential regulatory role played by 5´UTR and 3´UTR of the Oct4 gene in mouse BMSC and P19 cells.
Materials and Methods: The Oct4 5´UTR and 3´UTR sequences were cloned into pGL3 luciferase plasmid which led to the generation of pGL3 5´-UTR, pGL3 5´&3´-UTRs and pGL3 3´-UTR vectors. The vectors were transfected into BMSC and P19 cells followed by luciferase assay. 
Results: The assay of luciferase expression exhibited a direct link between the presence of Oct4 3´- UTR and the decrease of luciferase count in both cell lines; whereas 5´UTR indicated diverse behaviors in two cells. This discrepancy could be explained in view of the difference of cellular contexts in which the Oct4 UTRs act.
Conclusion: This study sheds some light on the role of UTR regions of mouse Oct4 in regulating post-transcriptional gene expression in pluripotent cells. These data represent potential to be used for the development of novel therapeutic approaches for a variety of malignancies.

Keywords


1. Xu N, Papagiannakopoulos T, Pan G, Thomson JA, Kosik KS, MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 2009; 137:647-658.

2. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 122:947-956.

3. Jiang J, Chan YS, Loh YH, Cai J, Tong GQ, Lim CA, et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 2008; 22:353-360.

4. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24:372-376.

5. Zaehres H, Lensch MW, Daheron L, Stewart SA, Itskovitz-Eldor J, Daley GQ. High-efficiency RNA interference in human embryonic stem cells. Stem Cells  2005; 23:299-305.

6. Scholer HR, Dressler GR, Balling R, Rohdewohld H, Gruss P. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J 1990; 9:2185-2195.

7. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998; 95:379-391.

8. Matin MM, Walsh JR, Gokhale PJ, Draper JS, Bahrami AR, Morton I, et al. Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells. Stem Cells 2004; 22:659-668.

9. Zeineddine D, Papadimou E, Chebli K, Gineste M, Liu J, Grey C, et al. Oct-3/4 dose dependently regulates specification of embryonic stem cells toward a cardiac lineage and early heart development. Dev Cell  2006; 11:535-546.

10. Tai MH, Chang CC, Kiupel M, Webster JD, Olson LK, Trosko JE. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 2005; 26:495-502.

11. Webster JD, Yuzbasiyan-Gurkan V, Trosko JE, Chang CC, Kiupel M. Expression of the embryonic transcription factor Oct4 in canine neoplasms: a potential marker for stem cell subpopulations in neoplasia. Vet Pathol  2007; 44:893-900.

12. Lengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S, Gokhale S, et al. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 2007; 1:403-415.

13. Trosko JE, Tai MH. Adult stem cell theory of the multi-stage, multi-mechanism theory of carcinogenesis: role of inflammation on the promotion of initiated stem cells. Contrib Microbiol 2006; 13:45-65.

14. Hatefi N, Nouraee N, Parvin M, Ziaee S-AM, Mowla SJ. Evaluating the expression of Oct4 as a prognostic tumor marker in bladder cancer. Iran J Basic Med Sci 2012; 15:1154.

15. Zangrossi S, Marabese M, Broggini M, Giordano R, D'Erasmo M, Montelatici E, et al. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker. Stem Cells 2007; 25:1675-1680.

16. Wilkie GS, Dickson KS, Gray NK. Regulation of mRNA translation by 5'- and 3'-UTR-binding factors. Trends Biochem Sci  2003; 28:182-188.

17. Makhanova N, Hagaman J, Kim HS, Smithies O. Salt-sensitive blood pressure in mice with increased expression of aldosterone synthase. Hypertension 2008; 51:134-140.

18. Ryan K, Bauer DL. Finishing touches: post-translational modification of protein factors involved in mammalian pre-mRNA 3' end formation. Int J Biochem Cell Biol 2008; 40:2384-2396.

19. Wickens M, Anderson P, Jackson RJ. Life and death in the cytoplasm: messages from the 3' end. Curr Opin Genet Dev 1997; 7:220-232.

20. Hu Z, Bruno AE. The Influence of 3'UTRs on MicroRNA function inferred from human SNP Data. Comp Funct Genomics 2011; 2011:910769.

21. Gray NK, Wickens M. Control of translation initiation in animals. Annu Rev Cell Dev Biol 1998; 14:399-458.

22. Mignone F, Gissi C, Liuni S, Pesole G. Untranslated regions of mRNAs. Genome Biol 2002; 3: REVIEWS0004.

23. Sangeeta C, Jayanta KP. Role of 5 - and 3 -untranslated regions of mRNAs in human diseases. Biol Cell 2009;? 251–262.

24. Krebsbach PH, Kuznetsov SA, Satomura K, Emmons RV, Rowe DW, Robey PG. Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts. Transplantation 1997; 63:1059-1069.

25. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 947–956.

26. Matoba R, Niwa H, Masui S, Ohtsuka S, Carter MG, Sharov AA, et al. Dissecting Oct3/4-regulated gene networks in embryonic stem cells by expression profiling. PLoS One 2006; 1:e26.

27. Lengner CJ, Welstead GG, Jaenisch R. The pluripotency regulator Oct4: a role in somatic stem cells? Cell Cycle  2008; 7:725-728.

28. Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development  1996; 122:881-894.

29. Solter D. Mammalian cloning: advances and limitations. Nat Rev Genet 2000; 1:199-207.

30. Izadpanah R, Trygg C, Patel B, Kriedt C, Dufour J, Gimble JM, et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 2006; 99:1285-1297.

31. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz
RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature  2002; 418:41-49.

32. Pesole G, Liuni S, Grillo G, Licciulli F, Mignone F, Gissi C, et al. UTRdb and UTRsite: specialized databases of sequences and functional elements of 5' and 3' untranslated regions of eukaryotic mRNAs. Update 2002. Nucleic Acids Res 2002; 30:335-340.

33. Pesole G, Grillo G, Larizza A, Liuni S. The untranslated regions of eukaryotic mRNAs: structure, function, evolution and bioinformatic tools for their analysis. Brief Bioinform 2000; 1:236-249.

34. Mazumder B, Seshadri V, Fox PL. Translational control by the 3'-UTR: the ends specify the means. Trends Biochem Sci  2003; 28:91-98.

35. Yi R, Poy MN, Stoffel M, Fuchs E. A skin microRNA promotes differentiation by repressing 'stemness'. Nature  2008; 452:225-229.

36. Tay Y, Zhang J, Thomson AM, Lim B, Rigoutsos I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 2008; 455:1124-1128.

37. Tay YM, Tam WL, Ang YS, Gaughwin PM, Yang H, Wang W, et al. MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells  2008; 26:17-29.

38. Orom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell  2008; 30:460-471.

39.http://www.umm.uniheidelberg.de/apps/zmf/mirwalk/predictedmirnagene.php.